This invention relates to the inhibition of the activity of tumor necrosis factor (TNF). TNF is known to have a causative role in many diseases such as chronic inflammation, certain types of arthritis, late stage wasting disease in cancer (cachexia), auto immune diseases such as lupus, and septic shock.
TNF is a cytokine which possesses multiple activities and affects almost every type of cell in the body. It was initially identified and named because of its ability to cause hemorragic necrosis of certain tumors in mice. It was subsequently recognized as the causative agent in the wasting condition in late stage cancer known as cachexia. A single gene codes for a membrane-bound 26 kD TNF protein; proteolytic cleavage produces circulating 17 kD TNF. Both are believed to be active. TNF is produced by most cells of the immune system, certain other cells and is constituitively produced in the thymus and by many leukemic cell lines.
TNF plays a critical role in the immune system and inflammation which may be ascribed to three different TNF functions. Evidence indicates that TNF is important in 1) facilitating leukocyte adhesion to and migration through endothelial cells by effects on remodeling of the extracellular matrix as well as on regulating cell adhesion molecules and integrins, 2) cell growth and differentiation and 3) programmed cell death (apoptosis) and cytotoxicity. The Immunopharmacology and Therapeutics section of the Department of Experimental Therapeutics (Grace Cancer Drug Center) has made a number of significant contributions toward clarifying the effects of TNF on host defense systems, and is currently using this information to develop effective anticancer therapies.
TNF's multiple activities may be explained in part by the existence of two receptors for TNF (molecular weights of 55 kD, TNFR1, and 75 kD, TNFR2). They are members of a TNFR superfamily which share considerable extracellular homology, indicating that they bind to similar ligands, but limited intracellular homology, suggesting that they may initiate different signal transduction/second messenger systems. Consistent with their assignment to this receptor superfamily, the TNFRs: 1) share extracellular homology and bind both TNF and lymphotoxin (TNF.beta.) with high affinity, and 2) have little intracellular homology, consistent with TNF's effects on a multitude of molecules involved with toxic/detoxification mechanisms and signal transduction. Recent evidence suggests that the cytoplasmic portions of the receptors bind different proteins, and that these proteins may also bind to other TNFR superfamily members.
The relative roles of TNFR1 and R2 signaling in TNF effects differ depending on the cells under investigation, and the effect examined. Both receptors are present on most cells, complicating the assignment of a specific effect to a specific TNFR type. Data indicate that TNFR1 stimulation, including receptor clustering, leads to cell lysis (both necrotic and apoptotic mechanisms have been reported); whether or not TNFR2 signaling contributes to this lysis is not always addressed. The "death domain", believed to signal lysis, at least in part through activation of the apoptotic pathway, is present in the cytoplasmic portion of both the TNFR1 and another TNFR superfamily member, Fas, whose binding also induces apoptosis. It is unclear if TNF and Fas induce apoptosis by the same mechanism. Several studies have linked activation of TNFR2, probably including clustering, to stimulation of proliferation, but TNFR2's role in TNF mediated lysis is less defined. It does appear that, under certain conditions, TNFR2 signaling and receptor clustering are responsible for a component of TNF mediated lysis.
A small number of transformed cell lines are sensitive to direct TNF cytotoxicity, but a greater number are TNF resistant. In the large majority of cases, TNF resistance of these cells does not appear to be correlated with a lack of TNF receptors or with defects in the internalization of the TNF-receptor complexes. In fact, many resistant cells are lysed by TNF when a protein synthesis inhibitor is present. This finding suggests that TNF induces/activates (at the mRNA level and/or the protein level) a protein or proteins which protect(s) the cell from TNF mediated lysis. TNF increases mRNAs for molecules related to cell adhesion, contact and substructure (ELAM-1/E-selectin, ICAM-1, VCAM-1, collagen, vimentin, collagenase, and stromelysin), certain cytokines and lymphokines (IL8 and MCAF/MCAP-1/murine JE), and several key transcription factors (p65 subunit of NF-kB, c-myc, c-jun, c-fos, and c-fra). A number of proteins/activities are known to be induced by TNF, although their respective mRNAs are not always found in TNF subtraction libraries. This suggests that TNF may also induce proteins at the translational/posttranslational level. TNF treatment of cells has been shown to result in increased secretion of a number of cytokines and immune factors (including IL6, IL8, IL1, CSF-1, GSCF and GMCSF) and receptors (MHC class I molecules, the p55 IL2 receptor and the EGF receptor).
Several proteins have been suggested, with varying numbers of supporting studies, as proteins which protect cells from TNF mediated lysis. However, their mechanisms of protection and their physiologic significance have yet to be elucidated. These proteins do not appear to protect the cells completely, and the investigators involved are in agreement that more than one protein is probably involved in protection of a given cell. These data are consistent with the hypothesis that multiple mechanisms contribute to TNF initiated lysis, and, therefore, the proteins which are protective to a cell correlate to the specific mechanism(s) by which that cell is lysed by TNF. Proteins which have been linked to protection from TNF induced lysis include: 1) manganese superoxide dismutase (MnSOD), an enzyme which converts toxic superoxide radicals to innocuous products, 2) plasminogen activator inhibitor 2 (PAI2), a cytosolic protein which is a member of the serpin family of protease inhibitors, 3) the .about.80 kD protein encoded by the A20 clone of a TNF subtraction library, 4) heat shock protein (hsp) 70 and hsp 27, and 5) endogenous TNF. Most of the studies implicating these proteins in resistance to TNF cytotoxicity involve transfection and expression of the protein in cells which then show increased resistance, or surveys which show increased levels of these proteins in subclones with increased resistance. However, levels of activity or mRNA of these proteins do not always correlate with the degree of resistance.
U.S. Pat. No. 5,136,021, entitled "TNF-Inhibitory Protein and a Method of Production", discloses a TNF inhibitory protein named TIP. Subsequent to filing the patent, the amino-terminal amino acid sequence (10 amino acids) of TIP was determined to be V-V-X-A-V-X-L-X-A-H (SEQ. ID. #1). As this protein was in very low abundance and the purification procedure was extremely labor intensive and difficult, it was not possible to obtain sufficient material for internal amino acid sequencing. These sequences were necessary to facilitate the search for the TIP gene. An alternative simpler and more effective method to isolate a "TIP" was therefore needed.